Rotavirus is the most common cause of severe, dehydrating diarrhea worldwide. Every year, over half a million children less than 5 years of age die due to rotavirus diarrhea. More than 80% of these rotavirus-related deaths occur in developing countries of sub-Saharan Africa and South Asia. Moreover, rotavirus is responsible for 25-50% of all diarrheal hospitalizations in both developing and developed countries and 23 million outpatient health care encounters annually in young children. Almost every child by 5 years of age will have been infected with rotavirus. Owing to the tremendous global burden of rotavirus disease, development of vaccines against this pathogen has been a priority for the past three decades. Two live, orally administered rotavirus vaccines available in the market are ROTATEQ® rotavirus vaccine (Merck and Co., Inc., PA, USA and Sanofi Pasteur MSD SNC, Lyon, France) and ROTARIX® rotavirus vaccine (GSK Biologicals, Rixensart, Belgium). In April and October 2009, WHO's Strategic Advisory Group of Experts (SAGE) reviewed additional efficacy data from different populations in Africa and Asia, and post-licensure studies in the America, and extended the recommendation for vaccination to all regions of the world.
The rotavirus capsid is made up of three concentric protein layers. The outer layer, consisting of VP7 and VP4, is lost during virus entry into the host cell. Rotavirus field isolates can be adapted to high-titer growth in tissue culture by treatment with trypsin and by supplementing the culture medium with trypsin, which cleaves VP4 into two fragments, VP8* and VP5*. It is known that protease inhibitors reduce the replication of rotavirus, in vitro and in vivo. When associated with triple-layer particles (TLPs), trypsin is inactive and not accessible to protease inhibitors, such as aprotinin. When the outer layer is solubilized by calcium-chelating agents, VP5*, VP8* and VP7 are released and the associated trypsin is activated, allowing cleavage of the viral capsid proteins, as well as other exogenous proteins. It is shown that addition of trypsin inhibitors significantly reduces synthesis of viral mRNA and viral proteins in cells and has a major inhibitory effect if present when virus enters the cell. This indicates that incorporation of trypsin into rotavirus particles may enhance its infectivity.
The infectivity is enhanced by treatment with trypsin. Trypsin converts a noninfectious fraction of virus into infectious virus by allowing this fraction to un-coat in the infected cell. Trypsin was found to cleave an 88,000-dalton structural polypeptide of bovine rotavirus generating 67,000- and 20,000-dalton cleavage products. Activation of Rotavirus with trypsin (Porcine origin) is established and addition of trypsin in the medium post infection was also done to get optimal yields. However, the source of trypsin is porcine i.e. from pigs. It has been shown that trypsin coming from porcine origin might contain other contaminating viruses like Porcine circovirus 1 and 2 which is not desirable. Traces of other risky pollutants like polychlorinated bi-phenyls (PCBs) can also be found when Porcine trypsin is used for growing rotaviruses. Therefore, it is essential that, trypsin of animal origin to be avoided from bioprocess of rotavirus culture, to the extent possible. Additionally, since porcine related products is also a barrier to certain religious faiths especially in the Islamic regions of the world, therefore, vaccines absolutely free of porcine origins will be more beneficial and useful. Process development studies were initiated to introduce the use of Recombinant trypsin which is novel step in the bioprocess to overcome the risk of PCB and other animal sources in the vaccine formulations. Recombinant trypsin is animal component free (ACF), highly specific in activity, free of contaminating chymotrypsin, free of adventitious agents of animal origin and safe for human consumption.
Wyeth had the earliest privilege to launch a commercial rotavirus vaccine in the market place in 1998-99. The composition of Wyeth's Rotashield included live human-monkey reassortant tetravalent rotavirus antigen, sucrose, monosodium glutamate, potassium monophosphate, potassium diphosphate, fetal-bovine serum, neomycin sulphate, in amphotericin B medium grown in fetal rhesus diploid cell lines. However, due to issues related with intussusception, and associated adverse effects following vaccine administration, Wyeth withdrew its commercial rotavirus vaccine formulations from market. Current vaccines, including one comprising a monovalent human serotype—P1A[8]G1 (Rotarix), and another comprising five human-bovine reassortants—G1, G2, G3, G4 and P1A[8] (RotaTeq), have demonstrated general efficacy and safety in clinical trials. Although considerable progress has been made with the association of intussusception with rotavirus vaccines, major unanswered questions still remain to be solved. It has been already observed and proved that available rotavirus vaccines generally have yielded poor efficacy when tested in developing countries which has led to concerns about the potential effectiveness of any future live oral rotavirus vaccine in these settings. (Bresee J S, Glass R I, Ivanoff B, Gentsch J. Current status and future priorities for rotavirus vaccine development, evaluation and implementation in developing countries. Vaccine 1999; 17: 2207-22). In high-income countries, few children die from rotavirus whereas, globally, most children who die each year from rotavirus infection are from low-income countries. Studies of rotavirus vaccines in low-income countries have shown moderate efficacy, unlike high income countries inspite of the vaccination programs. A combination of factors, may be attributed to such moderate results of rotavirus vaccines in the developing countries which includes limited access to healthcare (hydration therapy), poor infrastructure, higher costs of transportations of vaccines, special features in the mode of administration of the vaccine, specificity of serotypic coverage, along with a greater prevalence of malnutrition. Present research in this area necessitates further study of rotavirus vaccines in low-income countries and identify new ways to improve vaccine performance. Most importantly, the stability of the rotavirus vaccine formulations requires to be enhanced to overcome costly and long-term transportation and storage facilities in these low-income countries of interest. Although, the prior patents available in the field of rotavirus vaccines against rotavirus infections provide with experimental data for stability of the rotavirus vaccine formulations, practical implications on the present status of the art reflect major stability hurdles after the first year from date of manufacture of the vaccine. Therefore, effective stability studies are required to be shown for rotavirus vaccine formulations for at least 24 months of storage in order to ensure worldwide vaccination programs across least developed or other low-income countries wherein the fight against rotavirus may be considered to be still in its initial stage.
Rotavirus is an acid-labile virus having a half-life of less than 12 minutes at pH 2.0. It is rapidly inactivated at such acidic pH. The rotavirus vaccines are intended to be administered to the infants by the oral route, wherein the antigen in the rotavirus vaccine is most likely to be inactivated by stomach gastric acid. Therefore, to prevent inactivation of the virus by gastric acid, antacids or buffers are needed to be administered before or in combination with the oral rotavirus vaccination.
With U.S. Pat. No. 6,616,931 Merck, the first to enter in the domain of rotavirus vaccines. U.S. Pat. No. 6,616,931 claimed a rotavirus vaccine composition comprising a Liquid rotavirus vaccine formulation comprising a strain of rotavirus: (1×105 to 1000×105 pfu/ml), a sugar selected from the group consisting of: sucrose, lactose [1% to 70% w/v], mannitol, sorbitol, dextrose, fucose, trehalose, poly-aspartic acid, or N-acetylneuraminic acid, inositol-hexphosphate (phytic acid), and sialic acid, a phosphate [0.01M to 2M] selected from the group consisting of monophosphate, polyphosphate, phosphorylated sugars, and a carboxylate [0.05M to 2M] selected from the group consisting of succinate (0.05 M), citrate (0.07 M), fumarate, tartarate, maleate, lactate, recombinant Human Serum Albumin (0.5 to 1.25%), and polysorbate (0.1 to 2%) to liquid and lyophilized formulations. In case of lyophilized formulation nonionic surfactant selected from the group consisting of polysorbates, polysorbate80 (0.00 to 0.1%) is claimed. Citrate and Phosphates in the ranges of 0.05 M to 2 M and 0.01 M to 2 M have been used as buffering agents to counter the acidic environment of the stomach gastric acid in the rotavirus formulation. The commercialized vaccine “Rotateq” of Merck includes 5 live rotavirus strains (G1, G2, G3, G4, and P1), sucrose, sodium citrate, sodium phosphate monobasic monohydrate, sodium hydroxide, polysorbate 80 and fetal bovine serum. This commercially available rotavirus vaccine reflects the composition to its granted U.S. Pat. No. 6,616,931. The Rotateq vaccine had been made available in a 2 ml liquid solution for oral administration of 5 live human-bovine reassortant rotaviruses containing a minimum of 2.0-2.8×106 infectious units (IU) per reassortant dose, depending on the serotype, and not greater than 116×106 IU per aggregate dose.
However, U.S. Pat. No. 6,616,931 (WO2002/011540) also quotes that lyophillization of rotavirus vaccines can result in loss of viral titer during freeze-drying. This may result in low yields, and the potency of the lyophilized vaccine formulation as well is subject to be reduced below required levels resulting ineffective immunization. It also recognizes that prior reconstitution of a lyophilized formulation is subject to losing potency at room temperatures. Buffering agents for liquid formulations disclosed in this patent includes citrate, phosphate, succinate, bicarbonates and in combinations of other carboxylates such as fumarate, tartarate, lactate and maleate. Further, concentration of phosphate is taught to be kept lower than 0.35 M to avoid precipitation of the viral vaccine formulation. Use of bicarbonates for stabilizing the viral vaccine formulation is also expressly discouraged since it is taught to be detrimental to the stability of the formulation. Additionally, working examples of various buffer components except to those of citrates, succinates and phosphates are missing in the specification. Therefore, it seems that a need for novel buffer components exists for enhanced stability of the rotavirus vaccine and buffer liquid formulations.
Following Merck, Glaxo Smithkline also filed and subsequently granted U.S. Pat. No. 7,285,280 on rotavirus vaccine formulation. U.S. Pat. No. 7,285,280 claims a live attenuated rotavirus vaccine composition formulated with an organic antacid Sodium Citrate, or inorganic antacid Aluminium Hydroxide or Calcium Carbonate, and xanthane gum for oral administration. The vaccine formulation is lyophilized with Calcium Carbonate present as the antacid to be reconstituted with aqueous solution prior to administration. Alternatively, this patent also claims a vaccine formulation as a quick dissolving tablet in lyophilized form to be directly placed on the tongue of an infant/child wherein the rotavirus antigen, the antacid and xanthane gum are already present in the tablet. Accordingly, corresponding product prescription to U.S. Pat. No. 7,285,280 of commercially available “Rotarix”, of GSK is a lyophilized vaccine which needs to be reconstituted before administration by adding the diluent provided separately with the vaccine. “Rotarix powder and solvent for oral suspension” contains live attenuated human rotavirus RIX4414 strain produced on Vero cells not less than 106.0 CCID50, sucrose 9 mg and sorbitol 13.5 mg, dextran, Dulbecco's Modified Eagle Medium (DMEM), amino acids in the lyophilized powder whereas the reconstitution solvent includes, xanthan gum, calcium carbonate and sterile water. The vaccine dose after reconstitution is 1 ml and two separate doses are needed to be administered with a time interval of at least 4 weeks between the first and the second dose. Although a dosage formulation of 1 ml has been made available, it is not made available as an entirely stable single liquid formulation in practical terms. The vaccine formulation of Rotarix is lyophilized one, and comes with a separate oral applicator containing the diluent and a glass vial containing the lyophilized vaccine antigen in powder form along with a separate transfer adapter. A detailed 10 separate steps have to be chronologically followed in proper order to ensure proper reconstitution. This reconstitution of the vaccine by mixing vaccine and diluent has its own disadvantages. Administration of this vaccine undergoes a tedious process of first connecting the transfer adapter to the glass vial containing the vaccine antigen in powder form, followed by connecting the transfer adapter to the oral applicator, mixing the vaccine antigen and the diluent properly, ensuring the turbidity of the reconstituted solution, removing the transfer adapter, and then administering the vaccine. All these steps separately require efficient operative skills of the person/health care provider who is administering the vaccine to the infant/child/patient. Therefore it is still desired to have a single liquid formulation with appropriate dose volumes of rotavirus vaccine formulations.
Subsequently GSK's U.S. Pat. No. 8,192,747 claims a liquid oral rotavirus formulation comprising live attenuated rota virus antigen, a sugar (35% to 70% w/w) selected from sucrose, mannitol, maltose, dextrose, lactose and trehalose (40%-70%), a carboxylate which is sodium adipate (50 mM to 2 M restricted to 100 mM to 1 M further restricted to 400 to 700 mM and a carboxylic acid, Adipic Acid along with calcium ions, wherein the composition has an antacid capacity of at least 12 minutes. The carboxylates proved to have buffering capacity in this patent includes among others citrates, acetates, malonates, malates, glutamates, fumarates, lactobionates, maleates, glucouronates, galactouronates, galactarates, and tartarates along with corresponding acid and sodium hydroxide salt. Additionally, formulations disclosed in this patent also may include certain commercially available antacid components as well, preferably aluminium hydroxide and magnesium hydroxide, other water insoluble antacids mentioned are magnesium carbonate, aluminium carbonate, aluminium phosphate, mix of aluminium hydroxide and magnesium carbonate, aluminium magnesium hydrocarbonate, aluminium hydroxide-magnesium carbonate-sorbitol-mannitol, hydroxy-aluminium-sodium-carbonate, dihydroxy aluminium potassium carbonate. Although a list of antacids have been referred in this patent, stability of a vaccine formulation with a particular antacid has to be established. Further, antacid action on the live virus, while present as an ingredient to the vaccine formulation with other vaccine antigen and other stabilizers/preservatives or excipients is also questionable. Commercially available antacids mixed with vaccine formulations might cause the vaccine antigen to coagulate and form thick masses of the liquid formulation. Moreover, in liquid vaccine formulations, antacid remains much as a suspension and therefore not advisable to be included in the vaccine formulation. The product specification of commercially available next generation Rotarix vaccine of GSK provides a liquid vaccine formulation, “Rotarix oral suspension in pre-filled oral applicator” containing live attenuated Human rotavirus RIX4414 strain not less than 106.0 CCID50, produced in Vero cells, sucrose 1073 mg, Di-sodium Adipate, Dulbecco's Modified Eagle Medium (DMEM) and sterile water in a 1.5 ml dosage form to be administered orally.
Bharat Biotech International Limited owns a granted patent 242868 in India (PCT publication WO2007/132480) which also discloses unique rotavirus vaccine formulation which is stable at 2°-8° C. using citrate-phosphate buffers in the ranges of 100 mM to 150 mM and 310 mM to 400 mM respectively along with its other distinct formulation components. This patent involves separate administration of Citrate-Bicarbonate to the subject prior to the administration of the vaccine. According to this patent, Citrate-Bicarbonates are not included in the formulation itself. The present invention overcomes this drawback wherein the use of Citrate-Bicarbonate buffer system present in the vaccine formulation together with the rotavirus antigen, has been proved to retain the buffering capacity without affecting the vaccine stability of the formulation. Bharat Biotech's PCT publication WO2011/07363 has subsequently claimed and disclosed a novel bioprocess for preconditioning with Human Serum Albumin during virus-infected host cell propagation, thereby giving better characteristics of the vaccine stability.
As it is mentioned above, in rotavirus vaccine formulations, a particular buffer component is necessary to combat the gastric acid environment of the stomach. Although the initial GSK's commercial rotavirus vaccine formulations included antacids, but the latest commercial version of rotavirus vaccine of GSK has switched from antacids to buffers. In a simplistic manner, it could be stated that, an antacid is known to neutralize the pH of the stomach acidic environment, whereas a buffer retains the pH of the vaccine formulation itself in the highly acidic environment of the stomach. It is always advantageous to mix buffers into a formulation which can protect the vaccine antigen from gastric acid of the stomach, and also maintain vaccine stability as well.
A list of either buffering components, or antacids have been cited in the prior art for countering the gastric acid environment, the most preferred being citrate phosphate buffers. The current state of the art recognizes some inherent problems of using citrate phosphate buffers in vaccine formulations. They are the most physiological of the common buffers and mimic certain components of extracellular fluids, still they have a number of potential disadvantages. For instance, phosphates inhibit many enzymatic reactions including cleavage of DNA by restriction enzymes, ligation of DNA and bacterial transformation. Phosphates are more likely to become easily contaminated with micro-organisms and tend to precipitate during fixation. The US'931 patent family of Merck discourages use of Phosphates as a buffer above a certain limit of 0.35 M to avoid precipitation of phosphate salts in the vaccine formulations. The GSK's US'797 patent expressly mentions that the rotavirus vaccine formulations are preferably free of phosphates. Sodium Citrate has been used by GSK in its US'280 patent as an inorganic antacid. However, use of citrates by GSK in its subsequent US'797 as a buffer seems completely withdrawn. The US'797 claims carboxylates which is expressly intended to be derived from a di-carboxylic acid. Citrate Buffers are derived from citric acid. Citric acid has three carboxyl functional groups. Citrate buffers are prepared by mixing citric acid and preferably sodium citrate to get the desired pH. Therefore, citrate buffers are buffers prepared from tricarboxylic acids.
It is also evident from the background disclosure that, rotavirus vaccines require either antacids or buffers to withstand the highly acidic environment in the stomach. Buffers are important because, the suspended rotavirus antigen in a solution must not experience a loss of potency and should interact in a favourable manner with the rest of the excipients to optimise vaccine efficacy. They are crucial to the efficacy and stability of vaccines as dramatic increases or decreases in the pH of the solution can cause denaturation of the protein/rotavirus live antigen in the vaccine. The buffer to be used with the vaccine formulation requires the fulfilment of the criteria of being inexpensive and easy to prepare as well as resistant to oxidation to be stable. It is also important to choose a buffer that is not toxic to the preparation. A suitable buffer system should not reduce the titer of the antigen vaccine, as well as function as a potential ingredient to combat the highly acidic environment inside the patient to which the vaccine ought to be administered. Buffers being integral part in rotavirus vaccines, since they required to retain the pH of the vaccine formulation in the highly acidic pH of the stomach also needed to be ensured that the vaccine titer is not vitiated below its immunogenic levels. Buffers being another ingredient of rotavirus vaccine formulations intended to counter the acidic environment of stomach require establishing the stability of the vaccine formulation for its long-term use. Possibilities of use of other carboxylates as buffers might have been referred in the prior art, yet absolute and specific combinations of these buffers with antigen and other stabilizing excipients of the rota virus vaccine formulation is required to be established. Specific combination of appropriate buffers with antigen in one single vial, should not show any formation of precipitates, and must show long term stability. Despite an exhaustive list of many possible buffer components finds a mention in the prior arts of Merck and GSK, a biological composition cannot be accepted with mere assumptions and possibilities wherein the state of the art has not been supported by any experimental data. Merely Citrates, Phosphates, and to some extent Succinates have been experimentally shown in the prior art as the most widely acceptable buffers in vaccines.
Buffers are known to act in combination systems. Each combination buffer systems are specific to have its own unique physico-chemical properties and buffering capacity. Selection of a particular buffering combination system in a particular solution depends upon the kind of the composition. The present invention deals with rotavirus vaccine compositions which include live attenuated virus and a wide range of other formulation components such as one or more sugars, and partially hydrolysed proteins along with in presence of other possible stabilizing components. The components interact in a wide variety of ways with each other, and therefore, selection of a particular buffer-combination system that fits into the kind of a particular formulation thereby retaining its functional activity must be acknowledged. Contrary to possible buffers merely cited in the prior art, novel combination buffer systems which have not been cited in the prior art and their role in stabilizing the rotavirus vaccine formulations have been presented in details in the present invention, key feature being minimum of 2 years stability.
Therefore, novel vaccine compositions with novel buffers combination systems are invented for rotavirus vaccine formulations which are additionally safe and effective as well to ensure maximum rotavirus vaccine efficacy at the same time is also stable for comparatively higher amounts of time to effectuate better transportation of the rotavirus vaccines across the globe. The present invention proposes the use of novel buffer combination systems which are all absent in the prior art for rotavirus vaccine preparations capable of being readily ingested and assimilated by the human body without compromising on the viral titer and at the same time highly stable as well. Overcoming the existing limitations associated with rotavirus vaccine formulations with such ideal vaccine and buffer formulation(s) as described herein this invention will be of great advantage in worldwide vaccination against rotavirus infections.
Lyophilized vaccine formulations have extended stability but incur higher manufacturing costs. The logistics involved in case of lyophilized vaccines also decreases the chances of making rotavirus vaccines available in remote areas of the world. Sophisticated transportation facility for lyophilized vials adds on to the issue of logistics and increased expenditure of the vaccines. Therefore, inventors of the present invention have experimentally shown liquid rotavirus vaccine formulations with novel alternate buffers in a range of dose volumes including as low as 1 ml formulations to 2.5 ml dose volume formulations. for an extended period of up to at least 2 years. This is a considerable advancement over the current state of the art available on rotavirus vaccine formulations. Stable rotavirus vaccine formulations up to 2 years at refrigeration temperatures will facilitate easy transportation of the vaccines at much lower costs at the same time, maintaining the viral titer in the formulation to raise the required immunogenecity will also be made possible at ease.